The goal of this proposal is to optimize specific active immunotherapy for melanoma. Towards that end, several aspects of the immunology of human melanoma will be studied, to determine the mechanisms by which some patients achieve excellent clinical remissions with a therapeutic vaccine ("theraccine"). Theraccine has caused remissions of metastatic melanoma in 20% of patients, with a median response of 17 mo, associated with a rise in precursors of cytotoxic T lymphocytes (pCTL). Modifications of theraccine, such as lysates of 8-methoxypsoralen-sensitized UV light-irradiated cells, lyophilized lysates, and membrane vesicles, will be assessed by clinical trials, monitored closely by in vitro immunological assays. Low-dose cyclophosphamide and interleukin-2 will be combined with theraccine to try to increase CTL generation and thus, clinical response rates. A randomized Phase III trial in early stage melanoma (lb-lll) will be conducted in an attempt to eradicate microscopic residual diseases. To improve and simplify immunological correlations, additional assays will be compared with our estimate of the frequency of pCTL, including a proliferation assay and an assay for "lytic units" of cytotoxicity. Leukocytes and cytokines present in regressing lesions will be detected by immunohistochemistry. The antigens recognized by CTL and the interaction of CTL with melanomas will be studied in several ways. Specificity of tumor-infiltrating lymphocytes (TIL) and CTL from peripheral blood will be analyzed by cold target competition assays with melanoma lines and histologically different tumors. CTL clones will be established, phenotyped for surface markers and T cell receptor chains, and their specificity examined by direct cytotoxicity and cold target competition. CD8+ "classical" CTL and the broadly reactive (CD4+ only?) CTL found after theraccine treatment will be compared. The melanoma peptides recognized by CTL clones will be identified after Western immunoblotting by proliferation and competition assays. Immunogenic peptides will then be sequenced. The importance of accessory molecules and major histocompatibility complex alleles, especially HLA-A2, in the interaction of CTL and melanomas will be studied by statistical correlations with response, and by sorting or blocking of cytotoxicity by monoclonal antibodies in vitro. T cell receptor genes of cloned CTL from the blood or tumor and of uncloned TIL from regressing lesions will be examined with antibodies to gene products and by the polymerase chain reaction. Limited use of V region genes unique to melanoma will be identified by subtractive hybridization: of normal melanocytes and melanoma cells, and of squamous lung carcinoma and melanoma. Immunogenicity of fusion proteins will be tested by proliferation, competition assays, and cytotoxicity assays with protein- treated non-melanoma cells. A preselected combination of highly immunogenic MAA from cDNA clones will constitute the ultimate version of the melanoma theraccine.